Glucose and insulin responses following streptozotocin administration for the induction of a type 1 diabetes mellitus model in Microminipig

Abstract

We aimed to establish a streptozotocin-induced type 1 diabetes mellitus (DM) model in Microminipigs. Initially, a dose of 150 mg/kg streptozotocin was tested, and pigs exhibited characteristic triphasic patterns in glycemic responses: phase 1 (transient hyperglycemia), phase 2 (hypoglycemia), and phase 3 (sustained hyperglycemia). Blood glucose concentrations showed a transient increase in phase 1, and then dropped sharply in phase 2. Based on these findings, we conducted a follow-up study using a reduced streptozotocin dose of 125 mg/kg. This group exhibited glucose dynamics similar to those observed in the 150 mg/kg group, showing glucose reductions and mild lethargy. In phase 3, both groups developed sustained hyperglycemia with negligible insulin secretion, confirming the successful establishment of a type 1 DM model.

Diabetes mellitus (DM) is a major chronic disease affecting a large population worldwide [29]. In DM research, disease models induced by streptozotocin administration have been established in various animal species to meet various experimental objectives [6, 16]. Given the need for animals of a certain size in medical device development, pigs, whose metabolism closely resembles that of humans, have been used to develop streptozotocin-induced DM models [13].

The typical streptozotocin dosage for inducing type 1 DM in pigs ranges from 100–200 mg/kg body weight (BW), although susceptibility to streptozotocin varies depending on factors such as breed, strain, age, sex, diet, interindividual differences, circadian rhythm and routes of administration [2,3,4,5,6, 8, 11, 12, 14, 17, 18, 20, 22, 28]. However, although streptozotocin-induced β-cell destruction can result in life-threatening hypoglycemia, only a limited number of studies have reported detailed temporal profiles of glucose and insulin concentrations during the development of these pig models [11, 12, 22].

Administration of streptozotocin typically induces a triphasic glycemic response [1, 10, 11, 14, 22]. Phase 1, transient hyperglycemia, occurs immediately after streptozotocin injection and is primarily attributed to acute stress-induced hepatic glycogenolysis and inhibition of cell glucose uptake via glucose transporter type 2 (GLUT2) [7, 19, 24]. This is followed by hypoglycemia (phase 2), characterized by a rapid surge of insulin released from damaged pancreatic β-cells, which can often lead to severe hypoglycemic symptoms [5, 7, 9, 19, 21, 24, 27]. Irreversible β-cell necrosis and depletion then results in the cessation of insulin secretion, leading to sustained hyperglycemia (phase 3) and the establishment of a stable, insulin-dependent type 1 DM model [11, 12, 22]. Given that the hypoglycemic phase can occasionally result in life-threatening events, close and continuous monitoring of animals during Phase 2 is essential.

Among various porcine breeds, the Microminipig has gained attention due to its small body size and experimental suitability [15, 25]. Weighing less than 30 kg at maturity, it offers practical advantages as an experimental pig model. Its compact size reduces the labor required for long-term monitoring, making it a valuable model for applications such as the development of diabetes-related medical devices. In Microminipigs, a previous study indicated the induction of a type 1 DM model using a single dose of 250 mg/kg BW streptozotocin [26]. However, that study was conducted in a single animal and did not provide details on the induction process. To date, the optimal streptozotocin dosage required to reliably establish a type 1 DM model in Microminipigs remains unclear.

Therefore, in the present study, we aimed to identify an appropriate streptozotocin dosage for establishing a reliable type 1 DM model in Microminipigs. We administered 150 mg/kg BW of streptozotocin as a preliminary test to evaluate changes in blood glucose and insulin levels throughout the model induction period, based on previous studies in pigs [2,3,4, 11]. Based on these findings, we subsequently reduced the streptozotocin dose to 125 mg/kg BW and conducted a detailed investigation of post-administration glucose and insulin dynamics, along with histopathological analysis of the islets of Langerhans in the pancreas.

The study was approved by the Committee for Animal Research and Welfare of Gifu University (permit number: AG-P-N-20230154), and all animals were monitored by a veterinarian throughout the experiments. A total of four young adult male Microminipigs (7.5–8.8 months of age and 8.1–9.5 kg BW; Fuji Micra Inc., Fujinomiya, Japan) were used. The pigs were housed in individual pens under controlled environmental conditions: temperature of 21–24°C, relative humidity of 50–80%, and a 12:12-hr light-dark cycle. Animals were fed twice daily (8:00, 16:00) with 150–200 g/head of commercial feed (Multilac, Chubu Shiryo, Nagoya, Japan), and water was provided ad libitum.

Following a 24-hr fasting period, streptozotocin (No. 13104, Cayman Chemical Co., Ann Arbor, MI, USA), dissolved in citrate buffer, was slowly infused over 3 min into the jugular vein via a catheter (Arrow AK-04150-E-S, Teleflex Inc., Tokyo, Japan). There were two dosage groups (150 mg/kg BW and 125 mg/kg BW), with two pigs per group.

In the 150 mg/kg group, streptozotocin was administered at 16:00, and blood samples were collected every 2 hr for the first 24 hr after administration, and thereafter at 08:00 and 16:00 before feeding to measure insulin and blood glucose levels. In addition, as the pigs exhibited clinical signs of hypoglycemia, real-time blood glucose was monitored at bedside at intervals of 30 min to 2 hr from midnight until 24 hr after administration, at which time the pigs showed clinical recovery, using an analyzer employing the glucose dehydrogenase electrode method. During this period, when blood glucose levels fell below 30 mg/dL, 2–20 mL of 50% glucose (50% Glucose Injection Nisshin, Nissin Pharmaceutical Co., Ltd., Tendo, Japan) was administered via the catheter.

Based on observations of the 150 mg/kg group, hypoglycemia was expected to persist for ≤36 hr in the 125 mg/kg group. Therefore, blood samples were collected every 30 min for glucose measurements and every 2 hr for insulin measurements up to 36 hr post-administration, to closely monitor the phase 2 response.

After the streptozotocin induction, plasma insulin concentrations were determined using an ELISA kit (Mercodia Porcine Insulin ELISA 10-1200-01, Mercodia AB, Uppsala, Sweden). Euthanasia was carried out under deep anesthesia induced by thiopental sodium injection (RAVONAL 0.5 g for Injection, Nipro ES Pharma Co., Ltd., Settsu, Japan), followed by intravenous administration of potassium chloride (K.C.L. Drip Injection 15%, Maruishi Pharmaceutical Co., Ltd., Osaka, Japan). Necropsies were performed 6 days after streptozotocin administration in the 150 mg/kg group and 7 days after administration in the 125 mg/kg group, respectively.

The entire pancreas was collected from the four experimental pigs and three age-matched (11-month-old) male control pigs that had not been treated with streptozotocin. The organs were fixed in 10% neutral buffered formalin, routinely processed for paraffin embedding, sectioned at a thickness of 3–4 µm, and subjected to immunohistochemical staining using a mouse monoclonal antibody against porcine insulin (2d11–H5, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Histological evaluation was performed on sections from the head, body, and tail regions of the pancreas.

Both pig groups administered streptozotocin 150 mg/kg and 125 mg/kg developed typical diabetic symptoms, including polydipsia and polyphagia, following the onset of hyperglycemia (phase 3). During phase 1, whereas no vomiting was observed in the 150 mg/kg group, both pigs in the 125 mg/kg group vomited immediately after streptozotocin administration. During phase 2, the pigs in both 150 mg/kg and 125 mg/kg groups exhibited marked lethargy and unresponsiveness. These symptoms are consistent with the known effects of streptozotocin-induced hypoglycemia and were anticipated. Accordingly, a veterinarian specialized in laboratory animal medicine continuously monitored the pigs and provided appropriate supportive care throughout phase 2. All four pigs had good appetites during phase 3, despite experiencing BW loss. The weight loss was slightly greater in the 150 mg/kg group than in the 125 mg/kg group.

Figure 1 illustrates the changes in blood glucose and plasma insulin concentrations for the pig administered 150mg/kg and 125 mg/kg groups. The responses to streptozotocin treatment followed a triphasic pattern, consistent with previous studies [11, 14, 22]. Glucose concentration transiently increased immediately after streptozotocin administration. The duration of phase 1 was 8.0 hr in both pigs of the 150 mg/kg group, and 6.0 and 6.5 hr in the two pigs of the 125 mg/kg group. Plasma insulin concentrations remained low initially but began to increase thereafter. During phase 2, glucose concentrations fell to <40 mg/dL and remained at critically low levels in both groups. Plasma insulin levels increased transiently before declining again. In phase 3, glucose concentrations rose and remained persistently elevated at >250 mg/dL in both groups, while plasma insulin concentrations declined to near-zero levels.

Fig. 1.

Changes in blood glucose and plasma insulin concentrations in the 150 mg/kg and 125 mg/kg groups. Data are expressed as the mean of two pigs in each group. In both groups, glucose concentrations rose transiently immediately after streptozotocin administration and subsequently declined (phase 1). Glucose levels then remained at low concentrations (phase 2) before rising again to hyperglycemic levels (phase 3). Insulin concentrations remained low during phase 1, increased during phase 2, and declined to 0 mU/L in phase 3. In the 125 mg/kg group, spike-like elevations in glucose concentrations were observed in phase 2, corresponding to the periods immediately after glucose solution administration. In contrast, in the 150 mg/kg group, no distinct glucose spikes were detected despite 50% glucose solution being administered. Notably, insulin concentrations during phase 2 were substantially higher in the 150 mg/kg group compared with the 125 mg/kg group.

Table 1 summarizes the time course and concentrations of blood glucose and plasma insulin during each phase in 150 mg/kg and 125 mg/kg groups. First, in both pigs from the 150 mg/kg group, phase 1 lasted 8 hr. Blood glucose concentrations rose from baseline to peak levels of 207 and 175 mg/dL in each pig, respectively. Phase 2 began 8 hr after streptozotocin administration, during which blood glucose levels dropped to critically low values of 15 and 17 mg/dL, respectively. Plasma insulin concentrations peaked at 251 and 99 mU/L at 16 hr, respectively. In phase 3, the blood glucose concentrations increased and remained >250 mg/dL, while plasma insulin levels declined to 0 mU/L by 120 hr. Second, in the 125 mg/kg group, phase 1 lasted 6.0 and 6.5 hr in each pig, respectively. Blood glucose concentrations were comparable to those observed in the 150 mg/kg group, whereas plasma insulin levels were slightly lower. Phase 2 lasted 18.5 and 29.5 hr, with minimum blood glucose concentrations reaching 20 and 18 mg/dL, respectively. Peak plasma insulin concentrations were 37 and 41 mU/L at 14 and 16 hr, respectively. In phase 3, the blood glucose concentrations rose to hyperglycemic levels >250 mg/dL. Although insulin secretion was observed at the beginning of phase 3, plasma insulin concentrations declined to 0 mU/L by 120 hr.

Table 1. Summary of blood glucose and plasma insulin concentrations in each phase.

Dose of streptozotocin (mg/kg body weight)			150		125
Pig No.			4937	4938	4925	4944
Phase 1	Starting to ending time (hr after administration)		0 to 8.0	0 to 8.0	0 to 6.0	0 to 6.5
	Blood glucose concentration	Before administration (mg/dL)	60	88	79	86
		Maximum (mg/dL)	207	175	253	214
		Time to peak (hr after administration)	2.0	4.0	2.5	2.5
		At the end of Phase 1 (mg/dL)	170	96	50	66
	Plasma insulin concentration	Before administration (mU/L)	0	4.3	0	0
Phase 2	Starting to ending time (hr after administration)		8.0 to 24.0a)	8.0 to 24.0a)	6.0 to 24.5	6.5 to 36.0
	Duration (hr)		<40.0a)	<40.0a)	18.5	29.5
	Blood glucose concentration	At the beginning (mg/dL)	21	34	22	27
		Minimum concentration (mg/dL)	15	17	20	18
	Plasma insulin concentration	At the beginning (mU/L)	32	29	25	13
		Maximum (mU/L)	251	99	37	41
		Time to peak (hr after administration)	16.0	16.0	14.0	16.0
		At the end of Phase 2 (mU/L)	60	23	12	2
Phase 3	Starting time (hr after administration)		40.0	40.0	24.5	36.0
	Blood glucose concentration	At the beginning (mg/dL)	92	223	96	71
		Maximum concentration (mg/dL)	387	688	479	423
		Before necropsy (mg/dL)	253	439	345	388
	Plasma insulin concentration	At the beginning (mU/L)	19	12	10	3
		Before necropsy (mU/L)	0	0	0	0
	Body weight	Before administration (kg)	9.5	8.4	8.7	8.1
		At necropsy (kg)	8.7	7.5	8.1	7.6
	Weight loss (kg)		0.8	0.9	0.6	0.5

a) Because the clinical symptoms following streptozotocin administration in Microminipigs were initially unknown, we responded according to the symptoms observed after administering 150 mg/kg. Based on these observations, the pigs were provided with food and appeared to be clinically recovering; therefore, blood sampling was temporarily suspended after 24 hr.

Immunohistochemical analysis revealed marked islet atrophy [23], with very few insulin-positive cells detected in the 125 and 150 mg/kg groups. In contrast, insulin-positive cells were abundant in the non-diabetic pig (Fig. 2).

Fig. 2.

Immunostaining findings. Arrowheads indicate insulin-positive cells. A: Non-diabetic control (No. 4927), B and C: Nos. 4937 and 4938 in the 150 mg/kg group, respectively, D: No. 4944 in the 125 mg/kg group. Panels A, C, and D show histology of the pancreatic body, whereas panel B shows histology of the pancreatic head, as insulin-positive cells were absent in the pancreatic body but present in the pancreatic head. Numerous insulin-positive cells were observed in the non-diabetic control (A), whereas only a few (B, D) or none (C) were detected in streptozotocin-treated pigs. In another pig from the 125 mg/kg group (No. 4925), no insulin-positive cells were observed (data not shown). Scale bar: 100 μm.

This study demonstrated that administration of 125 and 150 mg/kg BW streptozotocin successfully induced type 1 DM in young adult Microminipigs. Both doses elicited a characteristic triphasic glycemic response and resulted in sustained hyperglycemia, accompanied by near-complete loss of insulin-positive cells in the pancreatic islets. Importantly, during phase 2, the pigs experienced potentially life-threatening hypoglycemia lasting up to 36 hr, underscoring the importance of careful monitoring during this vulnerable period.

The severity of streptozotocin-induced DM and associated mortality in pigs is known to be dose dependent [8, 12, 20]. While low doses (e.g., 35–85 mg/kg) generally cause only mild or reversible hyperglycemia, doses of ≥100 mg/kg are typically required to establish a stable type 1 DM phenotype. In our study, reducing the dose to 125 mg/kg BW produced outcomes comparable to 150 mg/kg while lowering the clinical burden. In particular, transient glucose spikes following administration of 50% glucose suggested that insulin-mediated hypoglycemia did not excessively contribute to symptom severity, and symptoms such as lethargy and unresponsiveness were notably milder. These findings support the selection of a lower, yet effective, dose to balance model stability and animal welfare.

Sensitivity to streptozotocin varies widely among species, breeds, and strains [11, 16, 20, 22]. Although pigs are typically more resistant than rodents or primates to streptozotocin-induced pancreatic β-cell destruction [6, 12], Microminipigs in this study responded to markedly lower doses (125–150 mg/kg) compared with other pig breeds. For example, Göttingen Minipigs and Large White pigs typically require 200 mg/kg for consistent model establishment [12, 20]. This heightened sensitivity highlights the importance of breed-specific dose optimization rather than applying a uniform protocol across pig models.

From a translational perspective, Microminipigs present several advantages as experimental animals. Their small body size, ease of handling, and compatibility with long-term monitoring make them particularly suitable for studies on metabolic diseases and implantable medical devices that require repeated sampling or intervention. By defining a dose that ensures both efficacy and safety, our findings provide a foundation for reproducible and ethically sound type 1 DM model development. However, further studies are warranted to determine whether this dose is equally effective in Microminipigs of different sexes, ages, or physiological states.

In conclusion, we demonstrated the feasibility of establishing a type 1 DM model in young adult Microminipigs using 125 mg/kg BW streptozotocin. This regimen achieved sustained hyperglycemia and near-complete β-cell depletion while reducing the risk of severe hypoglycemia, suggesting a practical platform for diabetes research. By clarifying the optimal dose and its potential applicability, this study provides an initial step toward future preclinical diabetes research using large-animal models.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.